Method and device for the measurement of chemical and/or biological samples

ABSTRACT

A device for the measurement of chemical and/or biological samples, in particular by means of luminescence spectroscopy, comprises an irradiation unit ( 12 ), a sample receiver ( 10 ), at least one optical unit ( 30 ) and a detector unit ( 40 ). Electromagnetic radiation of various wavelength ranges and/or polarizations are led from the sample ( 10 ) from the irradiation unit ( 12 ). The color marker in the sample ( 10 ), which contains at least one color marker, is stimulated into producing luminescence and gives off light. The emitted light is led by means of an optical unit to a detector unit. The light emitted by the color markers is detected by detectors ( 42, 44, 46, 48 ) in the detector unit. According to the invention, the measurement results may be improved on the irradiation unit ( 12 ) generating a pulsed irradiation. The irradiation unit is thus preferably controlled by a control unit ( 18 ) in such a way that the irradiation pulses impinge on the sample ( 10 ) in a temporal sequence.

The invention relates to a method and device for the measurement ofchemical and/or biological samples, in particular with the use ofluminescence spectroscopy.

A known method for the measurement of such samples is the screening ofthe samples. In screening, the interaction between two bio-molecules inthe presence of a test substance is studied. The bio-molecules can be,for example, the pairs of ligand-receptor, substrate-enzyme,protein-protein, or protein-DNA. A measurable signal may be producedeither by the bio-molecules themselves, or, as in most cases, samplemarkers have to be bound to the bio-molecules. As markers, substancesare employed that produce signals based on radio activity, luminescenceor absorption. When color markers are used that produce luminescence,the color markers are excited by electromagnetic radiation, e.g.suitable laser light, so that the electromagnetic radiation lifts anelectron to a higher energy level and the color marker gives off light,i.e. luminesces, by the electron returning to its original energy level.The probability of the return of the electron to its original energylevel, and thus of the emission of luminescence, is exponentiallydistributed overtime. The mean duration of the exited state is thereforealso referred to as the luminescence life. Since, in most cases,luminescent markers have but a slight influence on the interactionsbetween bio-molecules and are extremely sensitive compared to othermarkers, the use of luminescent color markers is particularlyadvantageous. Information on reactions between two bio-molecules areobtained by establishing a relation between the change in the lightemitted by the color markers and the reaction of the bio-molecules.

In an example of a measuring method, a target molecule is first exposedto a fluorescent reagent as a luminescent reagent adapted to bind to thetarget molecule. If, for example, the intensity of the fluorescencechanges during the binding, it can be used to quantify the binding. Inanother experiment, the target is exposed to both thefluorescence-marked reagent and a single substance. If a binding betweenthe substance and the target occurs, the fluorescence-marked reagent isseparated from the target. Thus, the ratio of bound and free markedmolecules changes. In turn, this entails a change in the emission offluorescence by the sample and it can be assessed whether and in how fara substance binds to the target.

In order to increase the amount of information to be acquired during ameasurement, a plurality of markers, in particular two color markers,may be used. Depending on the excitation energy of the two colormarkers, two electromagnetic radiation sources, e.g. lasers, ofdifferent wavelengths are employed to excite the color markers. Forexample, a red and a green color marker is used in combination with ared and a green laser. When red and green color markers were used, ithas been found that, when a red laser was used together with a greenlaser, the intensity of the light emitted by the red color marker isless than the intensity of the red color markers, when the same isirradiated exclusively with red laser light. This loss of intensityentails a loss of information and leads to falsified results.

This phenomenon can also be found when color markers of different colorsare used.

It is the object of the invention to provide a method and device for themeasurement of chemical and/or biological samples, in particular withthe use of luminescence spectroscopy or microscopy, by which improvedresults can be obtained. In particular, it is the object of theinvention, to achieve an improvement of the measured results forcommonly used red and green color markers.

The object is solved according to the invention with the method of claim1 and the device of claim 15, respectively.

According to the invention, an improvement of the measured results canbe achieved by pulsing the electromagnetic radiation used to test thesamples at least in a wavelength range and/or at least one polarization.A measuring volume of the sample including at least one color marker isirradiated with electromagnetic radiation of at least two differentwavelengths/wavelength ranges and/or polarizations, the radiation beingpulsed in at least one wave-length/wavelength range and/or at least onepolarization. Specifically, it is possible to provide for a relativemovement between color marker and measuring volume during a relativemovement time; in this case, it is desirable to pulse the radiation suchthat at least two radiation pulses impinge on the color marker perrelative movement time.

Pulsating one or a plurality of wavelength ranges and/or one or aplurality of polarizations, is preferably effected such that at leasttwo radiation pulses impinge on the color marker within a time duringwhich the marker diffuses through the measuring volume. The measuringvolume, which in high-throughput screening preferably corresponds to thefocal point of an irradiation optic, is preferably smaller than 10⁻⁹ l,in particular smaller than 10⁻¹² l. A particle, such as a molecule,provided with a color marker diffuses through the measuring volume.According to the invention, the time interval between successive pulsesis chosen such that a particle diffusing through the measuring volume ishit by at least two radiation pulses and the color marker is excited toluminesce at least twice. In this case, the relative movement timecorresponds to the diffusion time of the marker through the measuringvolume.

In another embodiment, a stationary marker (e.g. a marked immobilizedmacro-molecule, a marked sedimented or adherent cell, etc.) may beexamined, the relative movement being effected in particular by astepped movement of the measuring volume. This scanning process can beperformed either by moving the sample with the exciting beam path beingstationary or by scanning the sample by varying the position of theexciting beam path. In this case, the relative movement time correspondsto the time required to move the measuring volume along the marker or tomove the marker through the measuring volume by moving the sample. Thescanning could also be continuous, this embodiment eventuallycorresponding to a step-wise scanning in infinitesimally small steps.

Of course, any intermediate form is conceivable between these extremes,such as scanning slowly diffusing markers or using a parallelized opticconfiguration (e.g. by using a plurality of optical fibers (a fiberbundle)) to simultaneously produce several measuring volumes.

Typical diffusion times of particles through the measuring volume arewithin the range of 50 μs to 100 ms, in particular 2 ms to 10 ms. Theinterval between successive radiation pulses is preferably chosen asshort as to have the individual particles preferably be hit by at least100 radiation pulses even with diffusion times that short. It is mostpreferred to have at least 1,000 and in particular at least 5,000radiation pulses hit during the diffusion through the measuring value.Because of the high number of radiation pulses, the electrons of thecolor markers are raised to higher energy levels several times,therefore giving off photons several times while falling back to theiroriginal level. The more light is emitted from a color marker bound to aparticle, such as a molecule, the more information can be acquiredwithin the diffusion time.

This allows for an increase in the expressiveness of the informationobtained. Similarly, it is also desirable to provide a high number ofradiation pulses during the other above-mentioned possible relativemovement times. In one embodiment of the present method, such aradiation pulse could be sub-pulses as they are known from multi-photonexcitation, in particular dual-photon excitation, to excite aluminescence emission by the color marker.

When using electromagnetic radiation, for example, with two differentwave-length ranges, for example, such as red and green laser light, thered or green laser light continuously irradiates the sample and therespective other laser light is pulsed. When using red and green laserlight, preferably the green laser light is continuous and the red laserlight is pulsed. The luminescence signal caused by a pulsed excitationhas a characteristic course in time, the intensity decreasing over timeafter one excitation to the next excitation. The course in time of apulsed excitation is thus known with sufficient exactness. Theexcitation and the emission of the color markers excited by the otherwavelength range have a continuous course in time. Both luminescencesignals emitted by the sample are thus discernible by their differentcourses in time.

It is thus possible to significantly improve the measured results bypulsing a light source. Since laser light of different wavelengths isemitted and only one of both laser light sources has to be pulsed, whentwo laser light sources are used, this is a simple and economicmodification of the device required to practice the method.Specifically, it is advantageous in the above example to pulse the redlaser, since green pulsed lasers are still very costly today. Otherwise,a combination of all wavelengths tuned to the color markers to beexamined is possible, the use of at least two different wavelengthsbetween 350 nm and 800 nm being preferred. Moreover, in anotherembodiment of the present method, the use of UV laser diodes would bepossible. Another advantage of the present method is that only onedetector is needed to detect the two luminescence signals emitted by thesample.

Of course, the present invention can also be applied for more than twodifferent wavelength ranges. It is likewise possible to use differentpolarizations of the radiation exciting the color markers, instead ofusing different wavelength ranges. Similarly, an improvement of themeasured results can be achieved with the above present method if onlyone color marker is employed, since the same is excited differently byradiation of different wavelength ranges and/or different polarizations,for example, so that different sets of information can be obtained.

Among other things, the invention is based on the fact that a red colormarker excited by red laser light could be destroyed by green laserlight. The destruction is caused by an electron of the red color markeris raised to a higher energy level by the red laser light and is furtherexcited by the green laser light. This excitation by the green laserleads to an increase in the probability of the destruction of the redcolor marker, e.g. by ionization. In most cases this destruction isirreversible, i.e. the color marker can no longer be excited to emitluminescence and is lost for further measurement. This causes adeterioration in the measuring signal. This is also true for colormarkers of other colors.

In a particularly preferred manner, a further inventive improvement ofthe measuring signal can be achieved by pulsing the radiation used toexamine the samples, such as the laser light, and by usingelectromagnetic radiation having at least two different wavelengthranges and/or polarizations that are given off in a deferred manner.Here, the pulsing is effected in at least two wavelength ranges andpolarizations, respectively. The electromagnetic radiation of differentwavelength ranges and/or different polarizations thus impingessuccessively on the sample. For example, the risk of a destruction ofthe color marker is reduced by the fact that the radiation pulse thatcould destroy the color marker impinges at a moment in time in which ithighly probable that the excited electron has already returned to itsoriginal state.

By pulsing the electromagnetic radiation in all wavelength ranges and/orboth polarizations, further improvements of the measured results can beobtained.

An improvement of the measured results can be obtained by the aboveinventive method even if only a single color marker is present in thesample. This color marker is excited differently by the electromagneticradiation of different wavelength ranges and/or different polarizations.For example, different polarizations can be detected in the emittedradiation, allowing to draw conclusions on the rotational properties ofthe color markers.

The terms color substance marker/color marker/marker refer to both amarker supplied to the sample (such as rhodamine, oxazine, cyanine oranother color substance) and a marker inherent to the substance to beexamined, i.e. substances that preferably have luminescent properties,such as certain bio-polymers. The terms color substance marker/colormarker/marker also refer to other substances that may be examined usingspectroscopic or microscopic methods such as Raman scattering.Luminescence particularly also includes fluorescence andphosphorescence.

The term wavelength range used herein also includes, besides widerexcitation wavelength ranges typically extending over a plurality ofnanometers, more discrete wavelengths. In particular, it may be desiredto provide a monochromatic excitation for at least two differentwavelengths.

In the following, the invention will first be discussed with referenceto the use of electromagnetic radiation with different wavelengthranges. For a better understanding, this is done with reference to anexample including a red and a green color marker, each being excited byred and green laser light, respectively.

According to the invention, the laser light, i.e. the red and the greenlaser light in the example discussed, is pulsed to excite the colormarkers. In addition, the laser light pulses of the individualwavelength ranges are deferred in time relative to each other. Due tothe time-synchronized temporal offset of the pulses relative to eachother, the sample is always hit only by either a red or a green laserlight pulse at any moment. Even with an extremely small interval betweenthe green laser light pulse and the red laser light pulse, substantiallyfewer red color markers are destroyed. This increases the intensity orthe count rate of the light given off by the color markers. Thus, themeasuring results can be improved significantly.

According to the invention, when using red and green color markers,first, one or a plurality of light pulses of the red laser light and,subsequently, one or a plurality of light pulses of the green laserlight are directed to the sample. A time gap exists between the last redlight pulse and the first green light pulse. The time span is chosensuch that the excitation of the red color markers has substantiallydecayed again so that the electrons of the red color marker are notfurther excited from a high energy level by the green laser light pulseand the red color markers will not be destroyed in the course.

Preferably, in the present method, a laser light pulse is generated onlyafter the substantial decay of the excitation of the color markerexcited by previous laser light pulse of a different wavelength range.Thus, for example, the green laser light pulse is directed to the sampleonly when the excitation of the color markers excited by the red laserlight pulses has decayed substantially. Preferably, the next laser lightpulse is fired on the sample only hen the excitation of the previouslyexcited color marker has decayed by at least 90%, more preferably by atleast 95%, most preferably by at least 98%. Hereby, the measuringresults obtainable are significantly improved.

The necessary temporal offset of two successive light pulses ofdifferent wavelength ranges depends on the luminescence life of thecolor marker used. For a color marker with a life of 3 ns, the necessarytemporal offset is at least 2 ns, preferably at least 7 ns. For a lifeof 1 ns, it is at least 0.7 ns, preferably at least 2.3 ns. Pulses withdifferent intensities can be used, which is also true fornon-luminescent excitation. When a red and a green laser light are used,the distance between a green laser light pulse following a red laserlight pulse and a red pulse is crucial, since a green laser light pulseemitted too early or simultaneously with the red laser light pulse couldcause the destruction of the red color marker. However, the temporaloffset between a red laser light pulse following a green laser lightpulse and a green pulse is of no importance, since the green colormarker is not destroyed by the red laser light pulse. In this case, itshould merely be ensured that both pulses do not occur at the same time.

The pulse length of the individual radiation pulses impinging on themeasuring volume is preferably smaller than 1 ns. In particular, theradiation pulses are smaller than 500 ps and, more preferred, smallerthan 300 ps. The pulse length specifically depends on the time defermentof successive pulses. Here, as stated above, it has to be made sure,according to the invention, that the excitation of a color marker hassubstantially decayed. When modern lasers are employed, it is evenpossible to achieve pulse widths smaller than 10 ps.

For fluorescence excitation, the pulse frequency of the laser lightpreferably is 20-100 MHz, more preferably 60-80 MHz. The pulse frequencyof the individual laser light ranges are preferably identical so thatthe distances between the successive laser light pulses of differentwavelength ranges and/or polarizations remain constant over time.

Preferably, the sequence of the laser light pulses of the individualwavelength ranges and polarizations is repetitive. For example, whenthree lasers with different wavelength ranges are used, a light pulse issent to the sample first by the first, then by the second and thereafterby the third laser, whereafter the first laser again emits a pulse,followed by the second laser, and so on. When two lasers are used, e.g.a red and a green laser, the laser light pulses are preferably generatedalternately.

According to the above described preferred embodiments of the inventionwith reference to the use of laser light and the laser light pulsesgenerated according to the invention, the same inventive effect iscaused when other electromagnetic radiation, such as radiation in theinvisible wavelength range, is used. Corresponding radiation pulses havethe same effect as laser light pulses. Likewise, it is possible with theabove described embodiments, to use a sample with only one color marker.This one color marker is excited differently by radiation pulses ofdifferent wavelength ranges, the emitted radiation being different orpossibly being different depending on the wavelength ranges of theexciting radiation.

It is also possible to employ different pulse frequencies of commonmultiples (e.g. 40 MHz and 80 MHz). A constant offset in time betweenthe two laser light pulses would still be ensured. This is advantageous,if one of the two lasers, e.g. the red laser, has less power. Since theintensity of the luminescence emission is proportional to the excitationpower, a pulse frequency of the green laser (e.g. 40 MHz) reduced byhalf with respect to the red laser (e.g. 80 MHz) can result in acomparable intensity of the luminescence emission by the red and thegreen color marker. Despite the lower excitation power, the red colormarker is excited twice as often. In particular, the invention alsoprovides for detection with the use of Raman scattering instead ofluminescence effects (luminescence spectroscopy and luminescencemicroscopy). In this case, excitation sources with particularly highrepetition rates can be used because of the instantaneous emission ofthe Raman photons. Here, it is particularly advantageous, to exploitsurface-amplified Raman emission, since the effective cross section and,thus, the number of emitted photons is particularly high. Theconventional methods for exploiting the surface-amplified Ramanemission, such as the metallizing of surfaces, especially particlesurfaces, with silver, can be applied here.

The present method using two or more pulsed electromagnetic beams ofdifferent wavelength ranges, offers a plurality of possible applicationsin the field of transient spectroscopy, such as the transient absorptionspectroscopy (TRABS). For example, when using two pulsed laser lightsources, all color markers are excited by the first laser light pulse.The second, deferred laser light pulse selectively saturates orphoto-destroys certain color markers or fluorescent impurities. Hereby,a controlled lowering of fluorescence signals can be achieved andspecific color markers may be made preferred or discernible. It ispossible to employ a plurality of deferred pulses in differentwavelength ranges, instead of time pulses with different wavelengthranges, the respective wavelength ranges being selected such thatcertain color markers are saturated or photo-destroyed.

Results, corresponding to those obtained with the use of differentwavelength ranges, may also be obtained with different polarizations ofthe electromagnetic beams. For example, instead of using red and greenlasers, similar measuring results can be obtained with the use ofvertically and parallel polarized light that also allows to makestatements on the substance examined.

The wavelength range of green laser light used preferably ranges from480-550 nm, more preferred 485-535 nm. The wavelength range of a redlaser preferably ranges from 630-690 nm, more preferred 535-655 nm.Preferred possible green laser light sources are high-quality argon ionlasers with monochromatic excitation at 488 nm, 466 nm, 502 nm, 515 nm,528 nm, or Nd:YAG Lasers with monochromatic excitation at 492 nm or 532nm. Red laser light sources preferably are krypton lasers withmonochromatic excitation at 647 nm, such as red laser diodes availablefor various excitation wavelengths.

Often applied methods using two color markers with two lasers ofdifferent colors are coincidence analyses discussed here with referenceto fluorescence. Here, it can be determined in how far the color markersoccur simultaneously or separately, i.e. to what degree they are boundto a common reagent or to two separate reagents. This makes use of thefact that in case of a simultaneous occurrence the fluorescent light ofboth colors is detected at substantially the same time, while in case ofa separate occurrence, the detection of the fluorescent light of bothcolors is randomly distributed over time. Again, the example of red angreen may serve to explain this. A special case of coincidence analysisis the cross correlation analysis. Here, the fluctuations over time ofthe fluorescent light of one color marker, F_(green)(t), are registeredby a second detector detecting those of the other color markerF_(red)(t). The cross correlation function G(t_(c)) is calculated basedon these fluctuation traces. $\begin{matrix}{{G\left( t_{c} \right)} = \frac{< {{F_{green}(t)}{F_{red}\left( {t + t_{c}} \right)}} >}{< {F_{green}(t)} > < {F_{red}(t)} >}} & {{Eq}.\quad 1}\end{matrix}$(t: measuring time, t_(c): correlation time, < . . . >: averaging overt)

A cross correlation function not equal to zero will only be obtained, ifthe fluorescent lights of both color markers are connected(“correlated”) with respect to time. This is true when they are bound toa common reagent. The amplitude of the cross correlation function,G(t_(c)=0), allows for a direct statement on the concentration,C_(green+red), of this twice color-marked reagent compared to theconcentrations, C_(green) and C_(red), of the once marked reagents(C_(green) and C_(red) can be determined by other analytic methods).$\begin{matrix}{{G\left( {t_{c} = 0} \right)} = \frac{C_{{green} + {red}}}{\left( {C_{green} + C_{{green} + {red}}} \right)\left( {C_{red} + C_{{green} + {red}}} \right)}} & {{Eq}.\quad 2}\end{matrix}$

By the already mentioned destruction of the color markers, an undesiredreduction of the concentrations, C_(green+red), of the twice colormarked reagent (and of C_(red), the red marked reagent) and, thus, adecrease of the cross correlation amplitude, G(t_(c)=0) occurs. Such ananalysis of a biological system by cross correlation would lead tofalsified results and underestimate the actual biological concentrationof the twice color marked reagent, C_(green+red). Therefore, theinvention provides for a temporal offset between the red and the greenlaser light pulses. By preventing the red color marker from beingdestroyed, this disadvantageous influence on the correlation amplitudeis canceled out and a true cross correlation or coincidence analysis isobtained.

Another problem with measuring methods using two color markers with twolasers of different colors is the cross-talk of the luminescence signalof both color markers. This will be explained with reference tofluorescence. The absorption and emission spectra of fluorescent colorsare comparatively wide, i.e. they extend over a relatively largewavelength range, and they may overlap. This may cause the followingproblems that do not allow for an unambiguous attribution of thefluorescent light to a particular color marker or excitation laser—e.g.,they could lead to falsifications in the cross correlation function.These will again be explained with reference to red and green:

-   -   i) the fluorescent light of the red color marker may also be        excited by the green laser (in a restricted manner)—the red        fluorescent light excited by the green laser and the red laser,        respectively, overlap;    -   ii) a (small) part of the fluorescent light of the green color        marker overlaps with the red fluorescent light (“crosstalk”)—an        overlapping of the fluorescent light emitted by the green and        the red color markers occurs on the detector for the red        radiation;    -   iii) the fluorescent light of the red color marker may be        generated not only by the red laser, but also—through resonance        energy transfer—by the green color marker excited by the green        laser—similar to the case i), an overlapping of the red        fluorescent lights excited by the red laser and, indirectly        through energy transfer, by the by the green laser.

It is the intention of the invention, to prevent this crosstalk of thefluorescence signals by deferring the red and green laser light pulsesrelative to each other. The fluorescent light excited by the green andthe red laser can then be separated with respect to time and allows foran unambiguous attribution, as can be explained with reference to thethree problem cases: after the green laser light signal, only theportion of the green color marker (suppression of the “crosstalk”) or ofthe red color marker in the red fluorescent light, the red color markerbeing excited directly or indirectly by energy transfer. If, after decayof this fluorescence, the red laser light pulse follows, the redfluorescent light only contains portions of the red color marker exciteddirectly by the red laser. If, however, the green laser is timed to thedecay of this fluorescence, the fluorescent light can clearly beattributed to the color markers or the exciting lasers. Thus, anuncompromised cross correlation analysis becomes possible, for example.

A further inventive application of the temporal offset between two laserlight pulses is the detection of different polarizations of theluminescent light—in the following referred exclusively to fluorescentlight—of a color marker by only one detector. In conventional measuringmethods for detecting different polarizations of fluorescent light, thecolor marker is excited by a laser polarized in a certain plane X andthe polarization portion of the fluorescent light parallel and verticalto X is registered either simultaneously on two different detectors withthe help of a polarization beam splitter, or, separated in time (severalseconds), on a single detector by a timed switching of the transmissiondirection of a polarization filter. This allows to draw conclusions onthe rotational properties of the examined color markers. It is the ideaof this invention, to generate time-separated fluorescence signal pulses(decay pulses) of different polarization by two time-separated laserpulses polarized in the plane X and in the plane Y perpendicular thereto(this is possible even with only one laser when split). If the detectionis done on a single detector with a polarization filter with a constanttransmission direction, a comparable detection of the fluorescent lightcomponent parallel and vertical to the polarization plane of the lasermay be performed: the transmission direction of the polarization filteris assumed to b the plane X; if the first laser light pulse is emittedwith a polarization also in the plane X, the portion of the fluorescentlight polarized in parallel to the polarization plane of the laser isthen detected; after the decay of this fluorescence, the second laserpulse is emitted with a polarization in the plane Y vertical to theplane X; thereafter, only the portion of the fluorescent light that ispolarized perpendicular to the plane X of the laser is detected; afterits decay, another laser pulse is emitted with a polarization in theplane X; etc. This detection of the two polarization portions is donealmost simultaneously since the delay between the two laser light pulsesis restricted only to the decay of the fluorescent light which is withinthe range of the fluorescence life of the color markers, typicallybetween 1-4 ns. This “simultaneous” detection of both polarizations cantherefore be achieved with little material effort using only onedetector and one laser, so that it is of great interest for anyapplication employing fluorescence anisotropy and polarizationmeasurement.

In a similar manner, a delay between the red and the green laser allowsfor a “simultaneous” detection of the red and the green luminescencesignal on only one detector with a delay in the range of ns.

Thus, the present method allows for a two color cross-correlationanalysis with only one detector.

To obtain particularly good measuring results when using differentwavelength ranges and/or polarizations, it is preferred to employ ahigh-sensitive confocal microscope.

Another advantageous possible application of the present method lieswith analyses employing fluorescence resonance energy transfer (FRET).In FRET analyses of chemical and/or biological samples, advantage istaken of the fact that the excitation energy of a substance (donor),previously excited with a particular wavelength, can cause luminescenceof another substance (acceptor). The acceptor is thus additionally orexclusively excited by energy emitted from the donor (energy transfer).The effectiveness of this energy transfer is extremely dependent on thespatial distance and the spatial orientation of the donor and theacceptor, so that variations in the distance between these twosubstances can be studied very effectively using FRET.

Due to the primary excitation of the donor, the same could in principleemit luminescent light. However, this is attenuated or nonexistent(quenched) by the energy transfer and can thus no longer be detected.Immediate changes in the donor substance that would normally bediscovered immediately by its luminescent light can now be observed onlyindirectly through changes in the FERT effectiveness. As stated above,this depends from further factors (such as the donor-acceptor distance).The possibility to additionally observe the luminescent light from thedonor would allow a distinction between the various effects. If, whenpracticing the present method, first a sample is pulsed or continuouslyirradiated with light ot the acceptor excitation wavelength, asaturation of the corresponding acceptor color markers, i.e. anexcitation of most of those color markers, is obtained. A light pulse ofa second wavelength (time deferred with respect to the light of thefirst wavelength, if the same is pulsed) excites the donor color markerwhich in turn indirectly excites the acceptor color marker through FRET.Since many of the acceptor color markers have already been excited(saturated) by the light of the first wavelength, the FRET excitation bythe donor color marker is less. The inventive use of the pulsation ofone of both wavelength ranges or the delay of the pulses of bothwavelength ranges is advantageous in that less quenching of theluminescence of the donor color marker is effected. It is avoided that alarge portion of the luminescent light of the donor color marker isinvisible, since its excitation energy is used up by the FRET excitationof the acceptor color marker. By suitably selecting the temporal offsetor the power of the light of the first wavelength, a different extent ofsaturation of the acceptor color marker is obtained, and thus an optimumsuppression of the FRET quenching or the FRET signal is set.

A further possible application of the present method lies with animproved use of a FRET analysis employing FRET cascades. As indicatedabove, the effectiveness, and thus the observability of FRET, stronglydecreases with the distance between donor and acceptor and disappears ata maximum distance (typically about 100 nm). In FRET cascades, aplurality of color markers is provided in a sample, so that this maximumpossible distance can be increased. For example, a green color marker isexcited by a green laser as the first donor (donor 1). Through FRET, thesame excites a yellow color marker as the second donor (donor 2) which,through another FRET excitation, in turn excites a third, red colormarker as an acceptor to emit luminescence signals. Thus, the distancevariations between the donor 1 and the acceptor can be detected over aneven larger region, since the energy transfer from donor 1 to theacceptor passes over donor 2 (FRET cascade) and, for example, themaximum distance can be used twice. A disadvantage of the method thuspracticed is the insecurity whether a change in the acceptorluminescence signal has been caused by a change in the distance, andthus a FRET change, between donor 1 and donor 2, or by a change in thedistance, and thus a FRET change, between donor 2 and the acceptor.

Using two temporal offset green and yellow pulsed light sources excitingonly donor 1 or donor 2, respectively, the FRET effectivities and thedistances between donor 1 and donor 2 can selectively be analyzed by thegreen laser and, for the donor 2 and the acceptor, by the yellow laser.Such, possibly even longer, FRET cascades are thus substantiallyimproved by the use of electromagnetic radiation pulsed in at least onewavelength range.

In all fields of application of the invention, the possibility ofvarying the temporal offset of both laser pulses is advantageous. Inthis manner, a temporal offset optimal for the improvement of themeasuring results (e.g. less photo destruction, more optimalcross-correlation, better signal-noise ratio) can be found. Moreover, itbecomes possible, by systematic studies, i.e systematic changes in thetemporal offset in different measurements, to characterize the mutualeffects of processes and/or states (e.g. photo destruction, transientabsorption, excited state of the color marker).

The present device serves to generate electromagnetic radiation pulsedin at least one wavelength range and/or one polarization. Preferably, apulsating radiation is effected in at least two wavelength ranges and/orpolarizations. Specifically, the device is constructed such, accordingto the invention, that the radiation pulses of the individual wavelengthranges and/or polarizations are temporally offset with regard to eachother. Pulsating the at least one wavelength range and/or the at leastone polarization is preferably effected such that at least two radiationpulses impinge on the color marker within the diffusion period in whicha color marker diffuses through a measuring volume. The same is true forthe other relative movement periods described before in connection withthe method. The number of pulses and their length preferably correspondto the magnitudes described above with reference to the method. Such adevice comprises a irradiation unit, a sample receiver, a detector unitand at least one optic unit.

The sample receiver serves to hold a chemical and/or biochemical samplecontaining at least one color marker in order to perform luminescencespectroscopy.

The detector unit serves to detect the radiation emitted by the sample.With the help of the optic units, the radiation is directed from theirradiation unit to the sample receiver and/or the radiation from thesample is directed to the detector unit.

The present device may be configured such that the light is directedfrom the irradiation unit to the sample receiver and the radiation fromthe sample is directed to the detector unit using the same optic unit,the same being arranged above or below the sample receiver. It isfurther possible to design the device such that the light from theirradiation unit is directed to the sample receiver by an optic unitlocated above the sample receiver and the radiation emitted by thesample is directed to the detector unit using another optic unitarranged below the sample receiver.

The irradiation unit configured according to the invention generatesradiation in at least two different wavelength ranges and/or twodifferent polarizations. Preferably, a laser unit is used as theirradiation unit. Here, two or more lasers are used, each producinglaser light of another wavelength range and/or another polarization, atleast one of the lasers being operated in a pulsed manner. The laserlight pulses of the individual wavelength ranges and/or polarizationsare mutually offset with regard to time, if, as preferred, a pulsationof both wavelength ranges or polarizations is effected. Thereby, asdescribed above with reference to the present method, a significantlybetter measuring result is obtained.

Preferably, the laser unit comprises a control unit that is connectedwith one or all lasers. Here, a separate control unit may be providedfor each individual laser, the individual control units beinginterconnected through a common control. Preferably, a single controlunit is provided as a mode coupler for all lasers of the laser unit. Afirst laser is controlled through this control unit. The second and anyfurther laser is preferably connected to the single control unit via atrigger wire. Because of the signal propagation times that depend on thelength of the trigger wire, the laser light pulses, the laser lightpulses of the second and any further laser can be delayed automaticallywith respect to those of the first laser. Other practical possibilitiesto obtain a delay between the lasers are path length differences betweenthe different beam paths, or other electric components changing thesignal propagation times.

The control unit of the present invention can preferably be constructedas described above with respect to the present method, so that, forexample, a laser light pulse reaches the sample only when the excitationof a color marker, excited by the previous laser light pulse of adifferent wavelength range and/or another polarization, hassubstantially decayed. Further, the control unit allows for a control ofthe sequence of the individual lasers as well as of the time intervalsbetween the laser light pulses.

In a preferred embodiment of the detector unit, the detector unit onlycomprises one combination detector connected to an evaluating unit. Thissingle detector detects all luminescent light pulses given off by theindividual color markers in the sample. Since the luminescent lightpulses arrive at the detector with an offset in time, due to the delayof the radiation pulses exciting the color markers, the individualdetected values can be attributed by the evaluating unit to thecorresponding color markers and/or polarizations they come from. Bycombining the information on the sequence of the radiation pulses andthe sequence of the detector signals in the evaluating unit, it ispossible to sort the signals in red and green signals or parallel andperpendicularly polarized signals.

In another preferred embodiment, the detector unit comprises twodetectors, one detector being for detecting the light emitted from thered color markers, the other being for detecting the light emitted fromthe green color markers. This ensures that no falsification of theresults occurs due to errors by the evaluating unit. Further, theevaluating unit may be arranged in a simpler manner. When providingthree or more color markers, the corresponding number of detectors canbe provided.

In order to acquire additional information, the a polarizing beamsplitter may be arranged downstream of the detectors. A light beamcoming from a certain color marker is thus split into two beams ofdifferent polarization.

Further information on the sample, e.g. rotation properties, can betaken from these two beams. In particular, following the above describedmethod, each of these detectors can measure different colors insuccession. Thus, it is possible to measure more than one color in bothpolarizations using two detectors.

By gating, i.e. the synchronous turning off of a detector during a laserpulse, the measuring method may further be improved. Besides the alreadyknown suppression of prompt stray light and the resulting increase inthe signal-noise ratio, the detection of the luminescence emissioncaused by a laser pulse can be suppressed specifically. When using aplurality of pulsed lasers of different wavelength ranges and/ordifferent polarizations, this allows for a specific studying oftransient states and processes.

Preferably, the device is designed as a high-precision confocalmicroscope. Preferably, the detecting unit comprises electronics withwhich to perform cross-correlation measurement, for example. The use ofa confocal optical structure is preferred also because of the highresolution in the direction Z, i.e. along the optical axis, and of thegood signal-noise ratio. However, any non-confocal optical measuringsystems can be used for the practice of the present method.

When using the present device with differently polarized radiation andonly one detector, the irradiation unit is preferably modified asfollows:

-   (1) The radiation from a single non-polarized radiation source, such    as a non-polarized laser, is split into two beams by a beam splitter    which are polarized differently by a polarization filter and which    are later on united again. The delay between the two differently    polarized bemas is realized by path length differences in the two    beam paths.-   (2) The radiation from a single non-polarized radiation source, such    as a non-polarized laser, is split into two beams of different    polarization by a polarizing beam splitter and afterwards united    again. The delay between both differently polarized beams is    obtained by path length differences in both beam paths.-   (3) Generating two radiation pulses of different polarization using    a single non-polarized radiation source can also be achieved with a    fast rotating polarizing filter in a non-split beam path. Here, the    speed of rotation of the polarizing filter is adjusted to the pulse    frequency of the radiation source so that the individual radiation    pulses alternately have a different polarization direction.-   (4) Two polarized pulsed radiation sources of the same wavelength    ranges but opposite polarizations are tuned to each other or    triggered, as in the two-color approach, such that their pulses    reach the sample successively with a delay.

Whereas all embodiments of the device have the advantage that only asingle detector is required to register the differently polarizedluminescent light portions, the embodiments (1)-(3) even require only asingle pulsed radiation source.

The present invention, as described before with reference to the method,is advantageously developed especially by the suitable radiation device.

By coupling in two temporally offset radiation pulses of differentwavelength ranges, e.g. red and green, and by using suitable opticalfilters, luminescent light of different wavelength ranges, e.g. read andgreen, can be detected separately and almost “simultaneously” with onedetector, as described above. Thus, methods such as coincidence analysisor two-color cross-correlation measurement can be performed with only asingle detector. This is not possible according to prior art.

With the present invention and the present device, the followingmeasuring methods can be performed: spectrometry, multi-photonexcitation (e.g. in two photon operation), laser scanning excitation,near field spectroscopy, Raman and Rayleigh scattered lightapplications, FIDA (fluorescence intensity distribution analysis)applications, two-dimensional FIDA applications, coincidence analysisand fluorescence life measurements. One may also use parallel confocalsystems, such as Nipkow discs, line scanners, or PAM arrangements.Preferably, gated CCDs, CIDs, CMOSs or a plurality of CCDs, CIDs, orCMOSs are used that measure different signal portions through colorsplitters or polarizers.

With the present method it is also possible to measure changes in theconformation of a particle if the particle is marked with at least twomarkers and/or shows intrinsic luminescence at at least two locations(intrinsic markers). In particular, the particle is marked at definedlocations with exactly two molecules. Here, the excitation is effectedin alternating polarization. In an arrangement allowing the simultaneousdetection of two colors and two polarization directions (for example,four detectors measuring the sample volume at the same time), thepolarization of the two differently colored excitation pulses may berandomly polarized with respect to each other, as long as the ratio ofthe polarization directions is constant in time. This also includeschanging polarization directions, as long as they occur periodically orquasi-periodically. When excitation photons hit the markers in a definedpolarization direction, the absorption and thus the intensity of theemitted luminescence depends on the orientation of the markers withrespect to each other. This dependence shows in the relationship betweenthe respective detected polarizations. With fast rotating particles orparticle portions or markers, an arrangement with four detectors, sinceotherwise the contributions from the two markers are mixed.Specifically, it is thus possible to track bistable states of molecularrotation axes, such as cis-trans rearrangements or chair rearrangementsof ring structures. More complex applications of this method alsoinclude the breaking or the forming of cysteine bridges between proteinunits or folding leaflet arrangements. Specifically, the detectionsignal can be overlaid by luminescence life effects that can also beused for quantifying. In this application, it may be preferred to setthe concentration of the sample such that, on average, less than oneparticle is in the measuring volume. It may also be preferred to fix theparticles on a surface, for example, the surface of other particles, inparticular nano particles, or on a planar surface, in particular in theform of arrays.

The present method further allows for a measurement of coded beads onthe basis of particles referred to as “quantum dots” in prior art orsimilar particles. To this avail, different excitation wavelengths areemitted successively and the luminescence intensity or life of theparticles is measured. Since the particles described could be adjustedexactly with respect to their luminescence properties, i.e. theirexcitation and emission wavelengths, for example, through size andmaterial selection, it is possible by combining, e.g., three excitationcolors, three emission colors and three luminescence lives todifferentiate between two to the power of nine=512 particles. Thus, theparticles described can be used directly without having to buildcombinations thereof into beads as is necessary in prior art systems.

Again, it may be preferred to set the concentration of the sample suchthat, on average, less than one particle is in a measuring volume. Itmay also be preferred to fix the particles on a surface, such as on thatof other particles, in particular nano particles, or on a planarsurface, in particular in the form of arrays.

The present method and the present device are particularly suitable forpharmaceutic active substance search (screening), for identifying andcharacterizing pharmaceutically relevant substances and molecules, foridentifying analytes in diagnostic applications, for genome analysis orfor cleaning and concentrating substrates.

The following is a detailed description of the invention with referenceto test results and a preferred embodiment of the device. In thefigures:

FIG. 1 schematically illustrates a preferred embodiment of the presentdevice,

FIGS. 2-4 illustrate diagrams of the fluorescence intensities of apurely red color substance solution irradiated with a different lightpulse delay of a red and a green laser,

FIG. 5-7 illustrate diagrams of the fluorescence decay in a red and agreen detection channel in tests described with reference to FIGS. 2-4,and

FIG. 8 shows a diagram in which the graph of cross-correlationmeasurements is illustrated.

The preferred embodiment of the present device illustrated in FIG. 1comprises a sample receiver 10. This sample receiver 10 is schematicallyillustrated as a single container holding the sample to be examined. Thesample receiver may be micro- or nano-titer plates, for example. In theexample shown, a laser unit 12 serving as the irradiation unit comprisesan argon laser 14 generating green laser light with a wavelength of 496nm. A second laser 16 is a red laser diode, generating laser light witha wavelength of 635 nm. Both lasers 14, 16 are operated in a rapidpulsed mode. When using the device of FIG. 1 to carry out the testsdescribed in connection with FIGS. 2-8, the pulse frequency was 73 MHz.

The argon laser 14 is controlled through a mode coupler 18 connected tothe argon laser 14 via a wire 20. Through the mode coupler, an exactpulse frequency is generated. The mode coupler 18 is further connectedto the red laser diode 16 via a trigger wire 22. By providing the modecoupler as a common trigger unit, the pulse frequency of both lasers 14,16 is identical. Due to the length of the trigger wire 22, the pulsesgenerated by the two lasers 14, 16 are delayed with respect to eachother. The delay is due to the signal propagation time of the controlsignals from the mode coupler 18 to the laser 16.

The light beams emitted by the two lasers 14, 16 are combined by adichroitic beam splitter 24 so that they pass along an identical beampath.

However, since the laser pulses are delayed with respect to each other,no overlapping of the individual pulses occurs. The laser light bundledby the dichroitic beam splitter 24 is directed towards the samplereceiver 10 by a dichroitic mirror 26 and focused through an objective28 into the sample held in the sample receiver 10.

The objective 28 and the dichroitic mirror 26 are already parts of anotic unit 30. The optic unit 30 further comprises a tube lens 34 and apinhole diaphragm 36. The light emitted by the color markers containedin the sample passes through the objective 28, the dichroitic mirror 26and the tube lens 34 by which it is focused on the pinhole diaphragm 36.This is a typical arrangement of a confocal microscope where portions ofthe light are canceled out by the pinhole diaphragm 36.

In the embodiment of the device illustrated, a detector unit 40comprises four optical filters 32, four detectors 42, 44, 46, 48 as wellas a polarizing beam splitter 50 and an evaluating unit 52. The beamspassing the pinhole diaphragm 36 are split by the polarizing beamsplitter 50 into a beam 54 with parallel polarized light and a beam 56with perpendicularly polarized light. The beam 54 is split into twobeams 60, 62 by a dichroitic beam splitter 58, one of the beamsincluding the light given off by the red color marker and the other beamincluding the light given off by the green color marker.

Correspondingly, the other polarized beam 56 is split into a red and agreen beam 66, 68 by a second dichroitic beam splitter 64, which aredetected by the detectors 46 and 48, respectively. The optic filters 32filter out edge portions of the emitted light, for example, which do notcome from the color markers but, for example, from the material of thesample receiver 10. The beams 60, 62, 66, 68 detected by the detectors42, 44, 46, 48 are transformed into electric signals and transferred tothe evaluating unit 52 which typically is a PC adapted to the device.The evaluating unit determines the type of reaction that has occurred inthe sample.

Instead of directing the laser light to the sample and to direct thelight emitted from the sample to the detector unit using a single opticunit, two optic units may be used. The present device may be arrangedsuch that the light from the irradiation unit to the sample receiver andthe radiation emitted from the sample are directed to the detector unitusing the same optic unit which may be located above or below the samplereceiver. It is further possible to design the device such that thelight from the irradiation unit is directed to the sample receiver by anoptic unit located above the sample receiver, and that the radiationemitted from the sample is directed to the detector unit through anotheroptic unit arranged below the sample receiver.

Instead of the irradiation unit with two lasers 14, 16 illustrated inFIG. 1, a irradiation unit with only a single light source may be used.To establish beam paths with two different wavelength ranges, a beamsplitter is provided behind the light source, which decouples 50%, forexample, of the light generated from the beam path, irrespective of thefrequency of the light. This may be an inclined mirror, for example,that covers 50% of the beam path. Due to path length differences, adelay in time may be obtained between the two beam paths established.Here, only a single pulsed light source is required. To cause differentwavelength ranges in both beam paths, a unit for changing the wavelengthis provided in one of the beam paths. This may be a frequency doublingmeans or a frequency multiplier, for example. Further, an OPA may beprovided. This is a non-linear crystal causing a frequency shift.Likewise, a Raman shifter may be provided to shift the wavelength rangein one of the two beam paths.

A corresponding device with only a single light source may also be usedwhen two beam paths with different polarizations are to act on thesample. Again, the beam path generated by the light source is split anda delay in the pulsed single light source is caused by the path lengthdifferences. To make a polarization change in one of the beam paths, apolarization filter is provided, for example, in the beam path as a unitfor changing the polarization.

In the measurements depicted in FIGS. 2-7, a purely red color substancesolution has always been examined. This is the color substance cyanin 5(Cy5) dissolved in water in a concentration of 5 nM. The fluorescencelife of Cy5 in water is 0.7 ns.

FIGS. 2-4 each illustrate the count rate of the detector over time. Inall three tests, the sample was irradiated exclusively with the redlaser within the first 5 s, with the red and the green laser in the timebetween 5s and 15 s, and again exclusively with the red laser between 15and 20 s. in all three measurements, the frequency of the two pulsedlasers was 73 MHz.

In the measurement illustrated in FIG. 2, no pulse offset between thelasers was effected in range from 5-15 s win which both the red and thegreen laser were on. Thus, the read and green laser light pulses hit thesample and the red color marker simultaneously. It is obvious from thediagram (FIG. 2) that the count rate decreases largely in the range from5-15 s. in those ranges, where the green laser was not on, i.e. in therange from 0-5 s and the range from 15-20 s, the count rate issignificantly higher. This illustrates the destructive influence of thegreen laser on the red color markers.

In the measurement depicted in FIG. 3, a pulse offset between the redand the green laser of 2 ns was set in the time section from 5-15 s. thepulses from the green laser always occurred 2 ns after those of the redlaser, the two laser light pulses alternately hitting the sample. As isobvious from the diagram (FIG. 3), the count rate in the range from 5-15s is significantly higher than in the diagram of the measurement takenfirst (FIG. 2). Thus, even within a temporal offset of 2 ns, a certaindecay of the excitation of the red color marker has occurred (about 94%for Cy5), so that a significantly lower number of red color markers hasbeen destroyed by the green laser.

The effect of the present method is especially obvious from FIG. 4. Inthis measurement, a pulse offset of 10 ns was set in the range from 5-15s. Here, the diagram shows no deviation of the count rate for the singleranges. Thus, it may be assumed that even after 10 ns approximately allpreviously excited red color markers have returned to their originalstate.

In FIGS. 5-7, the count rate of a red and a green detection channel isdepicted over time. The detection channels are the correspondingdetectors in connection with the evaluating unit. FIG. 5 corresponds tothe measurement in FIG. 2, FIG. 6 corresponds to the measurement in FIG.3, and FIG. 7 corresponds to the measurement in FIG. 4. The solid linerepresents the fluorescence caused by the red laser, while the brokenline represents the fluorescence caused by the green laser. Themeasurements depicted in FIGS. 5-7 were made in the time from 5-15 s(FIGS. 2-4), i.e. when both lasers were on. In FIG. 5, there is no delaybetween the red and green laser light pulses, in FIG. 6, the delay is 2ns as in FIG. 3, and in FIG. 7, the delay is 10 ns as in FIG. 4.

FIGS. 5-7 clearly show the shift of the maximum fluorescence signals ofthe red and the green detection channel with respect to each other, dueto the pulse offset. With a pulse offset of 10 ns, the two detectionchannels can clearly be separated from each other. For example, thisallows for the use of a single detector for both color markers, since itis known at which moment light signals from which color marker reach thedetector.

The test illustrated in FIG. 8 is a measurement of a double-strandedoligo-nucleotide marked with Cy5 and rhodamine green, dissolved in waterin a concentration of about 1 nM. The oligo-nucleotide has a length of66 base pairs and prevents energy transfer because of the distancebetween the two color markers. A cross-correlation measurement of thered and green fluorescent light was performed. Again, the red and greenlasers were pulsed and adapted to be shifted in time. The lower curve isthe course of the cross-correlation for overlying laser light pulses,i.e. for laser light pulses without delay. In the upper curve, the laserlight pulses were mutually offset. From equation 2, the upper curveyields a concentration, C_(green+red)=1 nM, of twice markedoligo-nucleotides, whereas the reduced amplitude of the lower curveleads to a lesser concentration, C_(green+red)=0.6 nM. Due to the pulseoffset, no photo destruction occurs and the cross-correlation analysisleads to less falsified results.

1. A method for measuring chemical and/or biological samples by means ofspectroscopic or microscopic methods, in particular using luminescencespectroscopy, wherein: the sample including at least one marker in ameasuring volume is irradiated with electromagnetic radiation of atleast two different wavelength ranges and/or polarizations, the excitedmarker emits radiation in an emission wavelength range, and the emittedradiation is detected by at least one detector, characterized in thatthe electromagnetic radiation used to excite the marker is pulsed in atleast two different wavelength ranges and/or polarizations and theradiation pulses of the individual wavelength ranges and/orpolarizations impinge on the sample with a temporal offset.
 2. Themethod of claim 1, wherein a relative movement between the measuringvolume and the marker, in particular between the measuring volume and asingle marker molecule, occurs in a relative movement period and theelectromagnetic radiation used to excite the marker is pulsed in atleast one wavelength range and/or one polarization such that at leasttwo radiation pulses hit the marker within the relative movement periodin which the measuring volume contains the marker.
 3. The method ofclaim 1 or 2, wherein the marker, in particular a single markermolecule, diffuses through the measuring volume during a diffusionperiod and the electromagnetic radiation used to excite the marker ispulsed in at least one wavelength range and/or one polarization suchthat at least two radiation pulses hit the marker within the diffusionperiod.
 4. The method of claim 1 or 2, wherein the marker is stationaryand a relative movement between the measuring volume and the markeroccurs by moving the measuring volume and/or by moving the samplereceiver containing the marker.
 5. The method of one of claim 1, whereina radiation pulse is generated only after the excitation of the marker,excited by a previous radiation pulse of a different wavelength rangeand/or a different polarization, has substantially decayed.
 6. Themethod of one of claims 1-5, wherein the radiation of the individualwavelength ranges and/or polarizations is emitted in repetitivesequence.
 7. The method of one of claims 1-6, wherein the sampleincludes two markers, each marker being excited by one wavelength rangeand/or one polarization, respectively.
 8. The method of one of claims1-7, wherein a red and/or a green color marker are used together withred and/or green excitation light, the green excitation light has awavelength range preferably of 480 to 550 nm, more preferred of 485 to535 nm, and the red excitation light has a wavelength range preferablyof 630 to 690 nm, more preferred of 635 to 655 nm.
 9. Device formeasuring chemical and/or biological samples by means of spectroscopicor microscopic methods, in particular by luminescence spectroscopy,comprising a irradiation unit (12) for generating electromagneticradiation in at least two different wavelength ranges and/orpolarizations, a sample receiver (10) for holding the sample includingat least one marker, a detector unit (40) comprising at least onedetector (42-48) for detecting the radiation emitted by the sample, andat least one optic unit (30) directing the radiation from theirradiation unit (12) to a measuring volume in the sample receiver (10)and/or directing the radiation emitted by the sample to the detectorunit (40), characterized in that the irradiation unit (12) generatesradiation pulsed in at least two different wavelength ranges and/orpolarizations and the radiation pulses of the individual wavelengthranges and/or polarizations are temporally offset.
 10. The device ofclaim 9, wherein a relative movement between the measuring volume andthe marker, in particular between the measuring volume and a singlemarker molecule, occurs in a relative movement period and theirradiation unit generates radiation that is pulsed such that at leasttwo radiation pulses hit the marker within the relative movement periodin which the measuring volume contains the marker.
 11. The device ofclaim 9 or 10, wherein the marker, in particular a single markermolecule, diffuses through the measuring volume during a diffusionperiod and the irradiation unit (12) generates radiation pulsed in atleast one wavelength range and/or one polarization such that at leasttwo radiation pulses hit the marker within the diffusion period.
 12. Thedevice of one of claims 9-11, wherein it comprises means for moving themeasuring volume and/or the sample receiver containing the marker. 13.The device of at least one of claims 9-12, wherein the irradiation unitis connected to a control unit (18), preferably a mode coupler, forgenerating the radiation pulses.
 14. The device of claim 13, wherein thecontrol unit (18) controls the radiation pulses such that a radiationpulse is generated only after the excitation of the marker, excited by aprevious radiation pulse of a different wavelength range and/or adifferent polarization, has substantially decayed.
 15. The device ofclaims 13 or 14, wherein the control unit (18) controls the radiationpulses such that the radiation pulses of the individual wavelengthranges and/or polarizations are emitted in repetitive sequence.
 16. Thedevice of one of claims 9-15, wherein the irradiation unit (12)comprises at least two radiation sources, in particular lasers (14, 16),for generating different wavelength ranges and/or polarizations.
 17. Thedevice of claim 12, wherein the irradiation unit (12) comprises a commoncontrol unit (18) for all radiation sources (14, 16).
 18. The device ofclaim 17, wherein the control unit (18) is connected to the radiationsources (14, 16) through a respective trigger wire (20, 22), the timeinterval between the radiation pulses being defined by the length of thetrigger wires (20, 22).
 19. The device of one of claims 16-18, whereinthe irradiation unit (12) comprises two radiation sources (14, 16), oneradiation source (14) generating red light and the other (16) generatinggreen light, and wherein the green light has a wavelength rangepreferably of 480 to 550 nm, more preferred of 485 to 535 nm, and thered light has a wavelength range preferably of 630 to 690 nm, morepreferred of 635 to 655 nm.
 20. The device of one of claims 9-19,wherein the irradiation unit (12) comprises only one non-polarizedpulsed radiation source (14), the irradiation unit (12) additionallycomprising (a) a beam splitter and, in each beam path established, apolarizing filter as well as a component for combining the beam paths,or (b) a polarizing beam splitter for establishing two beam paths ofdifferent polarizations as well as a component for combining the beampaths, or (c) a rapidly rotating polarizing filter in an unsplit beampath.
 21. The device of one of claims 9-20, wherein the irradiation unit(12) comprises two polarized pulsed radiation sources (14, 16) of thesame wavelength range but of opposite polarization.
 22. The device ofone of claims 9-21, wherein the detector unit (40) comprises only onecombination detector connected to an evaluating unit (52), theevaluating unit (52) evaluating the radiation emitted by the at leastone marker separately due to the time interval.
 23. The device of one ofclaims 9-22, wherein the detector unit (40) comprises two detectors (42,44), in particular one for detecting the light emitted by red colormarkers and one for detecting light emitted by green color markers. 24.The device of one of claims 9-23, wherein the detector unit (40)comprises a beam splitter (50), arranged before the detectors (42-48),for generating two beams (54, 56) of different polarization and eachbeam (54, 56) is detected by at least one detector (42, 44, 46, 48). 25.The device of at least one of claims 9-24, wherein the marker, inparticular a single marker molecule, has a characteristic fading timeduring which only it emits radiation, and the irradiation unit (12)generates radiation that is pulsed in at least one wavelength rangeand/or polarization such that at least two radiation pulses hit themarker within the fading period.
 26. The method of at least one ofclaims 1-8, wherein the marker, in particular a single marker molecule,has a characteristic fading time during which only it emits radiation,and the electromagnetic radiation used to excite the marker is pulsed inat least one wavelength range and/or one polarization such that at leasttwo radiation pulses impinge on the marker within the fading period.